Bone marrow stromal stem cells (BMSCs) are multipotent progenitor cells that have great potential as an osteoinductive and osteogenic component for engineered bone tissue. Incorporation of these cells into clinically effective graft materials relies on the establishment of in vitro culture techniques that can direct expansion and preferential differentiation into the osteoblastic lineage. Successful techniques would lead to the creation of a bone-like tissue replete with osteoconductive and osteoinductive factors to stimulate host integration and remodeling. Building on our evidence that BMSCs are mechanosensitive and that flow stimulates phenotypic markers of osteoblastic maturation, we seek to understand how flow modulates phenotypic markers. The specific aims of this project involve systematic evaluation of three complementary components of a perfusion strategy. 1) Characterize the effect of shear stress magnitude and mass transport rate on expression of phenotypic markers of osteoblastic differentiation. 2) Assess the impact of intermittent flow strategies on osteoblastic differentiation. 3) Characterize the effect of adhesive ligand activity on differentiation and probe for synergy with shearing flow. The proposed studies - involving osteoprogenitor cells suitable for bone tissue engineering applications -will be performed under well-characterized conditions in a parallel-plate flow chamber to identify osteoinductive transport effects of perfusion, establish benchmarks of osteoblastic differentiation, and determine effective culture strategies. These findings will be used to predict optimal culture conditions for osteoblastic differentiation of BMSCs within perfused 3D scaffold geometries, with the goal of producing engineered bone tissues suitable for clinical testing.